Endoplasmic reticulum (ER) lumenal indicators in Drosophila reveal effects of HSP-related mutations on ER calcium dynamics
Endoplasmic reticulum (ER) lumenal indicators in Drosophila reveal effects of HSP-related mutations on ER calcium dynamics
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果蝇内质网 (ER) 管腔指标揭示 HSP 相关突变对 ER 钙动态的影响
DOI：
10.1101/2020.02.20.957696
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Oliva M
中科院分区：
文献类型：
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作者：
Oliva M
ER形成蛋白基因是遗传性痉挛性截瘫（HSP）中最常见的突变基因。这些基因在模式生物中的突变可导致ER网络的破坏。为了研究ER的生理作用如何受到这种破坏的影响，我们开发了一些工具来询问其Ca 2+信号传导功能。我们产生了GAL 4驱动的Ca 2+传感器，靶向ER腔，记录ER Ca 2+通量在identifiedDrosophilicons。UsingGAL 4线特定的Ib型或Is型幼虫运动神经元，我们比较了不同的内腔指标的反应，电刺激，轴突和突触前末梢。最有效的传感器ER-GCaMP 6 -210具有接近预期ER内腔浓度的Ca 2+亲和力。重复的神经刺激一般表现出短暂的增加，在轴突和突触前末梢的内腔Ca 2+。突变体缺乏神经元reticulon和REEP蛋白，同源的人HSP蛋白，表现出更大的ER内腔诱发反应相比，野生型，我们提出的机制，这种表型可能导致神经元功能障碍或变性。我们的线是有用的补充果蝇Ca 2+成像工具包，探索ER的生理作用，其在HSP和轴突变性的病理生理作用更广泛。
Genes for ER-shaping proteins are among the most commonly mutated in Hereditary Spastic Paraplegia (HSP). Mutation of these genes in model organisms can lead to disruption of the ER network. To investigate how the physiological roles of the ER might be affected by such disruption, we developed tools to interrogate its Ca2+signaling function. We generated GAL4-driven Ca2+sensors targeted to the ER lumen, to record ER Ca2+fluxes in identifiedDrosophilaneurons. UsingGAL4lines specific for Type Ib or Type Is larval motor neurons, we compared the responses of different lumenal indicators to electrical stimulation, in axons and presynaptic terminals. The most effective sensor, ER-GCaMP6-210, had a Ca2+affinity close to the expected ER lumenal concentration. Repetitive nerve stimulation generally showed a transient increase of lumenal Ca2+in both the axon and presynaptic terminals. Mutants lacking neuronal reticulon and REEP proteins, homologs of human HSP proteins, showed a larger ER lumenal evoked response compared to wild type; we propose mechanisms by which this phenotype could lead to neuronal dysfunction or degeneration. Our lines are useful additions to aDrosophilaCa2+imaging toolkit, to explore the physiological roles of ER, and its pathophysiological roles in HSP and in axon degeneration more broadly.